Specific Aims:[unreadable] Syntaphilin (SNPH) is a neuron-specific and axon-targeted protein initially identified in our lab as a candidate inhibitor of presynaptic function (1, 2). Transient expression of SNPH in hippocampal neurons and injection of SNPH into presynaptic SCG neurons in culture results in inhibition of neurotransmission. Our effort in generating SNPH KO mice has led to the discovery of a novel role for SNPH in the control of axonal mitochondrial motility (3), which may provide a molecular explanation for this inhibitory role in synaptic transmission following the over-expression of SNPH. Our ongoing study reveals that SNPH is required for maintaining a large number of axonal mitochondria in a stationary state through an interaction with the MT-based cytoskeleton. Three lines of evidence support this view. First, the mitochondria that associated with exogenously expressed GFP-SNPH were almost entirely immobile. This appears to occur through interactions with the cytoskeleton because SNPH contains a MT-binding domain, which is necessary and sufficient for SNPH-mediated immobilization of axonal mitochondria. Second, by recording mitochondrial movement in living neurons followed by retrospective immunostaining for endogenous SNPH, we demonstrate that the immobility of axonal mitochondria depends on their association with endogenous SNPH, and further reveal a binomial distribution with a strong correlation between the endogenous SNPH-tagged mitochondria (62%) and stationary mitochondria (65%). Finally, deletion of the snph gene in mice resulted in a substantially higher proportion of axonal mitochondria in the mobile state than that found in wild-type neurons, and reduced the densities of total and inter-bouton mitochondria in axons. The snph mutant neurons exhibit enhanced short-term facilitation during prolonged stimulation, by affecting calcium signaling at presynaptic boutons. This phenotype is fully rescued by reintroducing the snph gene into the mutant neurons. Thus, SNPH acts as a receptor for docking/retaining mitochondria in axons and at synapses. These findings reveal for the first time a neuron-specific protein capable of docking axonal mitochondria and regulating their densities within axons. [unreadable] [unreadable] Our study identifies a molecular link between the docking/retention of axonal mitochondria and activity-dependent synaptic plasticity. Presynaptic structure and function are highly plastic and undergo spontaneous and activity-dependent remodeling, thereby changing the demand for mitochondria in axons and at nerve terminals. In response to the diverse physiological states, mitochondrial balance between motile and stationary phase is a possible target of regulation by intracellular signals and synaptic activity. Thus, an important question remains to be addressed: How are motile mitochondria recruited to the SNPH-dependent stationary pool in response to neuronal activity and synaptic modification? Identification of SNPH as a docking protein provides a molecular target for such regulation. Future studies using the snph knockout mice model will provide molecular and cellular details on how SNPH regulates mitochondrial motility and presynaptic function in response to neuronal activity and signal transduction pathways, and demonstrate whether the snph mice are more susceptible to agents that cause motor neuron degeneration.[unreadable] [unreadable] Lab papers published related to the project:[unreadable] [unreadable] 1. Guifang Lao, Volker Scheuss, Claudia M. Gerwin, Qingning Su, Sumiko Mochida, Jens Rettig, and Zu-Hang Sheng (2000). Syntaphilin: a syntaxin-1 clamp that controls SNARE assembly. Neuron 25, 191-201.[unreadable] [unreadable] 2. Sunit Das, Judit Boczan, Claudia Gerwin, Philip Zald, and Zu-Hang Sheng (2003). Regional and developmental regulation of syntaphilin expression in the brain: a candidate molecular element of synaptic functional differentiation. Molecular Brain Research 116, 38-49.[unreadable] [unreadable] 3. Jian-Sheng Kang, Jin-Hua Tian, Philip Zald, Ping-Yue Pan, Cuiling Li, Chuxia Deng, and Zu-Hang Sheng. Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation. (Invited minor revision submitted to Cell).